Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage

Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia.     

Growth differentiation factor-9 stimulates rat theca-interstitial cell androgen biosynthesis

Growth differentiation factor-9 (GDF-9) was shown recently to be essential for early follicular development, including the appearance of the theca layer. Theca cells provide the androgen substrate for aromatization and estrogen production by granulosa cells. Using biologically active recombinant GDF-9 (rGDF-9) and an androgen-producing immortalized theca-interstitial cell (TIC) line or primary TIC, we have examined the action of this paracrine hormone on theca cell steroidogenesis. The effect of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells, GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived growth factor enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product, on TIC androgen production suggests a regulatory role of the oocyte on theca cell function and hence on follicle development and differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions on thecal cell recruitment and differentiation.     

Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development

The ovary contains a pool of primordial follicles containing oocytes arrested in meiosis that are the source of developing follicles for the female. Growth and differentiation factor-9 (GDF-9) is a member of the transforming growth factor beta superfamily of growth factors, and follicles of GDF-9 knockout mice arrest in the primary stage of development. The effect of GDF-9 treatment on the primordial to primary follicle transition and on subsequent follicle progression was examined using a rat ovary organ culture system. Ovaries from 4-day-old rats were cultured under serum-free conditions in the absence or presence of growth factors. GDF-9 treatment caused a decrease in the proportion of stage 1 early primary follicles and a concomitant increase in the proportion of stage 2 mature primary follicles. GDF-9 did not effect primordial follicles or stage 0 to stage 1 follicle transition. GDF-9 also did not influence stage 3 or 4 secondary follicle numbers. Isolated antral follicle granulosa and theca cell cultures were used to analyze the actions of GDF-9. GDF-9 treatment did not directly influence either granulosa or theca cell proliferation. The ability of GDF-9 to influence the expression of another growth factor was examined. GDF-9 treatment increased kit ligand (KL) mRNA expression in bovine granulosa cells after 2 days of culture. Ovaries from 4-day-old rats were also cultured with or without GDF-9 treatment, and total ovary expression of KL mRNA was increased by GDF-9. In summary, GDF-9 was found to promote the progression of early primary follicle development but did not influence primordial follicle development. The actions of GDF-9 on specific stages of follicle development may in part be mediated through altering the expression of KL.     

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.     

Molecular characterization of the follicle defects in the growth differentiation factor 9-deficient ovary.

Growth differentiation factor-9 (GDF-9), a secreted member of the transforming growth factor-beta superfamily, is expressed at high levels in the mammalian oocyte beginning at the type 3a primary follicle stage. We have previously demonstrated that GDF-9-deficient female mice are infertile because of an early block in folliculogenesis at the type 3b primary follicle stage. To address the molecular defects that result from the absence of GDF-9, we have analyzed the expression of several important ovarian marker genes. The major findings of our studies are as follows: 1) There are no detectable signals around GDF-9-deficient follicles for several theca cell layer markers [i.e. 17alpha-hydroxylase, LH receptor (LHR), and c-kit, the receptor for kit ligand]. This demonstrates that in the absence of GDF-9, the follicles are incompetent to emit a signal that recruits theca cell precursors to surround the follicle; 2) The primary follicles of GDF-9-deficient mice demonstrate an up-regulation of kit ligand and inhibin-alpha. This suggests that these two important secreted growth factors, expressed in the granulosa cells, may be directly regulated in a paracrine fashion by GDF-9. Up-regulation of kit ligand, via signaling through c-kit on the oocyte, may be directly involved in the increased size of GDF-9-deficient oocytes and the eventual demise of the oocyte; 3) After loss of the oocyte, the cells of the GDF-9-deficient follicles remain in a steroidogenic cluster that histologically resembles small corpora lutea. However, at the molecular level, these cells are positive for both luteal markers (e.g. LHR and P-450 side chain cleavage) and nonluteal markers (e.g. inhibin alpha and P-450 aromatase). This demonstrates that initially the presence of the oocyte prevents the expression of luteinized markers, but that the absence of GDF-9 at an early timepoint alters the differentiation program of the granulosa cells; and 4) As demonstrated by staining with either proliferating cell nuclear antigen (PCNA) or Ki-67 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) labeling, the granulosa cells of GDF-9-deficient type 3b primary follicles fail to proliferate but also fail to undergo cell death. This suggests that granulosa cells of type 3b follicles require GDF-9 for continued growth and also to become competent to undergo apoptosis, possibly through a differentiation event Thus, these studies have enlightened us as to the paracrine roles of GDF-9 as well as the normal steps of granulosa cell and theca cell growth and differentiation within ovarian follicles.     

Knockout of pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility

The ovulatory process is tightly regulated by endocrine as well as paracrine factors. In the periovulatory period, extensive remodeling of the follicle wall occurs to allow the extrusion of the oocyte and accompanying cumulus granulosa cells. Growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) are secreted members of the TGFbeta superfamily that are expressed beginning in the oocyte of small primary follicles and through ovulation. Besides its critical role as a growth and differentiation factor during early folliculogenesis, GDF-9 also acts as a paracrine factor to regulate several key events in preovulatory follicles. By analyzing GDF-9-regulated expression profiles using gene chip technology, we identified TNF-induced protein 6 (Tnfip6) and pentraxin 3 (Ptx3 or PTX3) as novel factors induced by GDF-9 in granulosa cells of preovulatory follicles. Whereas Tnfip6 is induced in all granulosa cells by the LH surge, Ptx3 expression in the ovary is specifically observed after the LH surge in the cumulus granulosa cells adjacent to the oocyte. PTX3 is a member of the pentraxin family of secreted proteins, induced in several tissues by inflammatory signals. To define PTX3 function during ovulation, we generated knockout mice lacking the Ptx3 gene. Homozygous null (Ptx3(-/-)) mice develop normally and do not show any gross abnormalities. Whereas Ptx3(-/-) males are fertile, Ptx3(-/-) females are subfertile due to defects in the integrity of the cumulus cell-oocyte complex that are reminiscent of Bmp15(-/-)Gdf9(+/-) double mutant and BMP type IB receptor mutant mice. These studies demonstrate that PTX3 plays important roles in cumulus cell-oocyte interaction in the periovulatory period as a downstream protein in the GDF-9 signal transduction cascade.     

Paracrine actions of growth differentiation factor-9 in the mammalian ovary.

Although the transforming growth factor-beta (TGF-beta) superfamily is the largest family of secreted growth factors, surprisingly few downstream target genes in their signaling pathways have been identified. Likewise, the identities of oocyte-derived secreted factors, which regulate important oocyte-somatic cell interactions, remain largely unknown. For example, oocytes are known to secrete paracrine growth factor(s) which are necessary for cumulus expansion, induction of hyaluronic acid synthesis, and suppression of LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated that absence of the TGF-beta family member, growth differentiation factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary follicle stage leading to infertility. In the present study, we demonstrate that mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles. To explore the biological functions of GDF-9 in the later stages of folliculogenesis and cumulus expansion, we produced mature, glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary cell expression system. A granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semiquantitative RT-PCR. We find that recombinant GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppresses urokinase plasminogen activator (uPA) and LHR mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increases granulosa cell progesterone synthesis in the absence of FSH. Since induction of HAS2 and suppression of the protease uPA in cumulus cells are key events in the production of the hyaluronic acid-rich extracellular matrix which is produced during cumulus expansion, we determined whether GDF-9 could mimic this process. Using oocytectomized cumulus cell-oocyte complexes, we show that recombinant GDF-9 induces cumulus expansion in vitro. These studies demonstrate that GDF-9 can bind to receptors on granulosa cells to regulate the expression of a number of gene products. Thus, in addition to playing a critical function as a growth and differentiation factor during early folliculogenesis, GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte microenvironment, processes which are essential for normal ovulation, fertilization, and female reproduction.     

Follicle-restricted compartmentalization of transforming growth factor beta superfamily ligands in the feline ovary

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Throughout reproductive life, follicle fate is balanced between growth and apoptosis. These opposing forces are controlled by numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor beta (TGFbeta) superfamily. TGFbeta, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor 9 (GDF-9) are present in the ovary of many animals; however, no comprehensive analysis of the localization of each ligand or its receptors and intracellular signaling molecules during folliculogenesis has been done. The domestic cat is an ideal model for studying ovarian follicle dynamics due to an abundance of all follicle populations, including primordial stage, and the amount of readily available tissue following routine animal spaying. Additionally, knowledge of the factors involved in feline follicular development could make an important impact on in vitro maturation/in vitro fertilization (IVM/IVF) success for endangered feline species. Thus, the presence and position of TGFbeta superfamily members within the feline ovary have been evaluated in all stages of follicular development by immunolocalization. The cat inhibin alpha subunit protein is present in all follicle stages but increases in intensity within the mural granulosa cells in large antral follicles. The inhibin betaA and betaB subunit proteins, in addition to the activin type I (ActRIB) and activin type II receptor (ActRIIB), are produced in primordial and primary follicle granulosa cells. Additionally, inhibin betaA subunit is detected in the theca cells from secondary through large antral follicle size classes. GDF-9 is restricted to the oocyte of preantral and antral follicles, whereas the type II BMP receptor (BMP-RII) protein is predominantly localized to primordial- and primary-stage follicles. TGFbeta1, 2, and 3 ligand immunoreactivity is observed in both small and large follicles, whereas the TGFbeta type II receptor (TGFbeta RII) is detected in the oocyte and granulosa cells of antral follicles. The intracellular signaling proteins Smad2 and Smad4 are present in the granulosa cell cytoplasm of all follicle size classes. Smad3 is detected in the granulosa cell nucleus, the oocyte, and the theca cell nucleus of all follicle size classes. These data suggest that the complete activin signal transduction pathway is present in small follicles and that large follicles primarily produce the inhibins. Our data also suggest that TGFbeta ligands and receptors are colocalized to large antral follicles. Taken together, the ligands, receptors, and signaling proteins for the TGFbeta superfamily are present at distinct points throughout feline folliculogenesis, suggesting discrete roles for each of these ligands during follicle maturation.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Transforming growth factor-beta(1) regulates differentiation of porcine granulosa cells in vitro

Transforming growth factor-beta (TGF-beta) is a potential regulator of ovarian function and follicular development. It is speculated that TGF-beta mediates the events in the follicle which culminate in ovulation of the oocyte. The complex processes which ultimately leads to this natural phenomenon must involve interactions between the 2 major follicular cell types, theca and granulosa cells, and the oocyte. Furthermore, a complex local regulatory system must exist to determine which follicles should undergo development and, eventually, which of those should ovulate or undergo atresia. To begin to understand this perplexing process, we must first understand the variables which control the function of each individual cell type. This study investigated the effect of TGF-beta(1) on FSH-induced porcine granulosa cell differentiation in vitro. Transforming growth factor-beta(1) was shown to inhibit progesterone production at high concentrations (0.1 and 10.0 ng/ml) after 12-, 24- and 48-hour treatment. However, TGF-beta(1) produced a biphasic effect on FSH-induced progesterone production during the 12-hour interval between the 36- and 48- hour treatment periods; TGF-beta(1) stimulated progesterone production at a low concentration (0.001 ng/ml) and inhibited production at high concentrations (0.1 and 10.0 ng/ml). The results obtained from the biphasic effect were not observed during any of the other incubation periods or intervals investigated. These results show that TGF-beta(1) has opposing effects on the differentiation of porcine granulosa cells as compared with those on rat granulosa cells. Moreover, TGF-beta(1) can produce opposing effects within the porcine granulosa cell itself which are specific to the concentration and treatment period used. The results of this study seem to suggest that TGF-beta(1) is species- and time-specific in its regulatory actions on FSH-induced porcine granulosa cell differentiation.     

Control of proliferation and differentiation of ovine granulosa cells by insulin-like growth factor-I and follicle-stimulating hormone in vitro.

Granulosa cells of antral follicles both proliferate and undergo differentiation. The aim of the present work was to study the mechanisms controlling the balance between proliferation and differentiation in granulosa cells during the development of antral follicles in the ewe. For this purpose, the responses of both activities to insulin-like growth factor-I (IGF-I) and to FSH in vitro were studied comparatively in granulosa cells from small antral follicles (1-3 mm in diameter) and large antral follicles (5-7 mm in diameter). In granulosa cells from large follicles, IGF-I enhanced both basal and FSH-induced progesterone secretion after a 24-h delay period; this effect was lower and further delayed in cells from small follicles. Reciprocally, FSH increased IGF-I-stimulated progesterone secretion in cells from large follicles. IGF-I increased the thymidine labeling index of granulosa cells from small follicles within 24 h and enhanced cell multiplication. In cells from large follicles, this effect was lower and delayed, but IGF-I also enhanced cell survival. Culture at high density of plating inhibited the proliferative response of both types of cells to IGF-I. FSH was without effect on granulosa cell multiplication. These results suggest that the cytodifferentiative and the growth-promoting effects of IGF-I are clearly distinct. We propose that they would be exerted on two distinct granulosa cell subpopulations, nonproliferating and proliferating cells, respectively. Differences in the responsiveness of cells from small and large follicles could be related to differences in the proportion of these two cellular subtypes.     

Mouse antral oocytes regulate preantral granulosa cell ability to stimulate oocyte growth in vitro

In this study we evaluated whether mouse oocytes derived from early antral or preovulatory follicles could affect the ability of preantral granulosa cells to sustain oocyte growth in vitro. We found that early antral oocytes with a diameter > or =75 microm did not grow any further during 3 days of culture on preantral granulosa cell monolayers in vitro, while most of the oocytes with a smaller diameter increased significantly in size. Similarly, about 65% of growing oocytes isolated from preantral follicles grew when cultured on preantral granulosa cells. By coculturing with growing oocytes fully grown early antral or preovulatory oocytes, a small proportion (about 10%) of growing oocytes increased in diameter, and changes in granulosa cell morphology were observed. Such effects occurred as a function of the fully grown oocyte number seeded and were not associated with a decrease in coupling index values. By avoiding physical contact between antral oocytes and granulosa cells, the proportion of growing oocytes undergoing a significant increase in diameter was about 36%. These results indicate that fully grown mouse oocytes can control preantral granulosa cell growth-promoting activity through the production of a soluble factor(s) and the maintenance of functional communications with surrounding granulosa cells.     

Actions of epidermal growth factor receptor/mitogen-activated protein kinase and protein kinase C signaling in granulosa cells from Gallus gallus are dependent upon stage of differentiation

Studies in both mammalian and nonmammalian ovarian model systems have demonstrated that activation of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways modulates steroid biosynthesis during follicle development, yet the collective evidence for facilitory versus inhibitory roles of these pathways is inconsistent. The present studies in the hen ovary describe the changing role of MAPK and PKC signaling in the regulation of steroidogenic acute regulatory protein (STAR) expression and progesterone production in undifferentiated granulosa cells collected from prehierarchal follicles prior to follicle selection versus differentiated granulosa from preovulatory follicles subsequent to selection. Treatment of undifferentiated granulosa cells with a selective epidermal growth factor receptor (EGFR) and ERBB4 receptor tyrosine kinase inhibitor (AG1478) both augments FSH receptor (Fshr) mRNA expression and initiates progesterone production. Conversely, selective inhibitors of both EGFR/ERBB4 and MAPK activity attenuate steroidogenesis in differentiated granulosa cells subsequent to follicle selection. In addition, inhibition of PKC signaling with GF109203X augments FSH-induced Fshr mRNA plus STAR protein expression and initiates progesterone synthesis in undifferentiated granulosa cells, but inhibits both gonadotropin-induced STAR expression and progesterone production in differentiated granulosa. Granulosa cells from the most recently selected (9- to 12-mm) follicle represent a stage of transition as inhibition of MAPK signaling promotes, while inhibition of PKC signaling blocks gonadotropin-induced progesterone production. Collectively, these data describe stage-of-development-related changes in cell signaling whereby the differentiation-inhibiting actions of MAPK and PKC signaling in prehierarchal follicle granulosa cells undergo a transition at the time of follicle selection to become obligatory for gonadotropin-stimulated progesterone production in differentiated granulosa from preovulatory follicles.     

Growth differentiation factor-9 and stem cell factor promote primordial follicle formation in the hamster: modulation by follicle-stimulating hormone

Growth differentiation factor-9 (GDF-9) and stem cell factor (SCF) influence follicle formation beyond the primary stage; however, factors influencing the formation of primordial follicles remain elusive. To determine whether GDF-9 and SCF promoted primordial follicle formation during ovarian morphogenesis in the hamster, and whether FSH had any modulatory influence, fetal ovaries were collected on Gestation Day 15 from pregnant hamsters treated with or without an FSH antiserum on Gestation Day 12 and cultured in vitro up to Day 9 with SCF, GDF-9, or FSH. The percentages and diameters of primordial, primary, and secondary follicles and their oocytes were determined by morphometric evaluation, and the expression of GDF-9 was detected by immunolocalization. SCF, GDF-9, and FSH promoted primordial and primary follicle formation, but GDF-9 was more efficient. The diameters of the follicles developed under GDF-9 or FSH, but not SCF, compared well with those developed in vivo. FSH- and GDF-9-induced folliculogenesis was attenuated by the SCF antibody. Similarly, in vitro formation of primordial follicles decreased markedly in ovaries exposed to the FSH antiserum in utero, which was reversed by SCF, GDF-9, or FSH; however, GDF-9 had a profound effect on follicular development. GDF-9 protein appeared exclusively in the oocytes on Postnatal Day 4; however, it appeared in vitro by 48 h, and the expression was upregulated by FSH. These results suggest that although SCF-induced primordial follicle formation constitutes primarily somatic cell development, GDF-9 influences both the oocyte and its companion somatic cells. FSH plays an important role in primordial folliculogenesis in the hamster via GDF-9 and SCF.